| Apple(Malus pumila)is one of the most economically important fruit crops grown worldwide.China is the country with the largest cultivated area and production of apple.Apple mosaic disease is the most common plant disease caused by viral pathogen,which occurs widely and severely in apple-produced areas and has been a serious threat to the development of apple industry.Accurate identification of pathogen is particularly important for the prevention and control of disease,but there is no agreement on the causal agent of apple mosaic disease in China[(outside of China typically associated with infection by the ilarvirus,Apple mosaic virus(ApMV].Recently,a novel ilarvirus,Apple necrotic mosaic virus(ApNMV),was isolated from apple trees with mosaic and necrotic symptoms in Japan.Then the survey of occurrence and distribution of ApNMV was performed in China,the association with mosaic symptoms was analyzed.Also,genome characteristics,taxonomic status,detection assays and pathogenicity of ApNMV were studied.The results in this study are as follows:A survey has been conducted,and a total of 291 apple mosaic leaves were collected from the major apple-growing provinces and regions(Shandong,Shaanxi,Shanxi,Gansu,Xinjiang and Beijing)in China.The mosaic symptoms in apple trees varied from mild,moderate to severe chlorosis,pale yellow mosaic to bright cream-colored irregular spots,rings.Bands and/or line patterns along the main veins were observed with uneven distribution on apple leaves.Sometimes,brownish necrotic spots were also observed.A very high percentage(i.e.,92.1%,268 out of 291)of symptomatic trees being infected with ApNMV was detected by RT-PCR and nested RT-PCR assays,but not with ApMV and PNRSV detected using special primer sets by RT-PCR.And we also analysed specificity of the primers used to be detected ApMV and PNRSV in apple mosaic samples.It demonstrated that ApNMV might be the main causal agent of apple mosaic disease in China,rather than ApMV or PNRSV.The complete nucleotide sequences of the six ApNMV isolates were obtained,ranged in size from 3378-3380 nt(RNA1),2778-2786 nt(RNA2)and 1909-1955 nt(RNA3),respectively.The similarities of Chinese ApNMV isolates of RNAs 1-3 genome sequences with the Japanese isolates P129(accession number:LC108993;LC108994;LC108995)were 95.4-96.5%(RNA1),88.5-94.5%(RNA2),and 86.2-91.1%(RNA3),respectively.The 5' untranslated regions(UTR)of RNA1 and RNA2 shared 95.1%and 84.8%nucleotide identities,respectively,and that of RNA3 showed a wide variation with only 74.1%nucleotide identities.But the 3' UTR shared 91.9-99.2%nucleotide identities over the 3'-most 125 nt.A highly conserved stem-loop structure was predicted and the two AUGC motifs were present at the base of stems in the 3' UTR.The intergenic region(IR)between MP and CP genes was found to be of 98-103 nt,which showed a large difference in length from that(145 nt)described in P129.ApNMV belonged to subgroup 3 of genus Ilarvirus of family Bromoviridae with typical genomic characteristics,as shown by phylogenetic trees constructed using the neighbor-joining method at both nucleotide and amino acid levels,which revealed homologies with ApMV and PNRSV.RNAs 1-3 segments of six Chinese ApNMV isolates were 49.2-59.8%identical to ApMV and 57.4-64.3%identical to PNRSV at nucleic acid level,respectively.And further comparative analysis of ApNMV with ApMV and PNRSV with all available MP and CP nucleotide sequences in GenBank suggested that ApNMV was distantly related to ApMV and PNRSV at evolutionary relationship.The recombinant plasmid to express ApNMV CP protein in prokaryotic cells was constructed.After a large amount of induced expression,the precipitated portion of the E.coli lysate was subjected to protein purification,and 2.4 mg of recombinant protein was copurified.And then polyclonal antibodies of ApNMV were prepared.A low-cost and high-efficiency ApNMV ELISA serological detection system suitable for large samples was established.Additionally,we constructed a recombinant plasmid for the transcription of ApNMV CP in vitro,and the RT-qPCR standard curve was made.A simple,specific,sensitive and reproducible one-step real time RT-qPCR assay was developed successfully for rapid detection and quantification of ApNMV in apple trees.To our knowledeg,it is the first report of ELISA and RT-qPCR detection techniques of ApNMV in China.The full-length infectious cDNA clones of ApNMV were constructed.The biological activity of the infectious cDNA clones was verified on N.benthamiana inoculated via agrobacterium-mediated method and sap inoculation,but no obvious symptoms were observed on inoculated N.benthamiana.ApNMV-infectious cDNA clones were also inoculated to cucumber(cv.'Suyo')cotyledons and significant mosaic symptoms in non-inoculated leaves were observed at 12 days postinoculation(dpi).The cucumber leaves infected with ApNMV initially exhibited yellow spots and gradually spread to form yellow chlorotic symptoms.Shrinking symptoms were also observed on the cucumber leaves.On the other hand,non-inoculated leaves of cucumbers inoculated by ApMV infectious cDNA clones exhibited obvious mosaic and chlorosis symptoms with slight leaf curling at 8 dpi.Slowly,the leaves infected with ApMV were severely yellow,chlorotic and curled,and the plants were dwarfed and stopped growing,eventually led to the death.In conclusion,we demonstrated a strong association of ApNMV with the mosaic disease of apple trees in China.Moreover,the complete nucleotide sequences of six Chinese ApNMV isolates were obtained,the taxonomic status was identified,two routine detection systems were established,and the pathogenicity of full-length ApNMV clones was verified.This study can provide an academic basis for investigation of the disciplinarian of the pathogenic prevalence,research on pathogenicity mechanism genetic engineering for disease resistance.It will be helpful to screen the apple genetic resources resistant to ApNMV and propose a reasonable prevention and control strategy for apple mosaic disease in China... |
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